MOLECULAR REGULATION OF ENDOTHELIAL NA K 2CL COTRANSPORT

本研究的目的是确定血管内皮细胞中Na-K-2Cl协转运体(NKCCl)的分子调控。这种转运蛋白被细胞收缩激活并迅速恢复细胞体积，从而在最小化内皮单分子层间隙形成从而维持血-组织屏障的完整性方面发挥潜在的重要作用。转运蛋白也被生长因子激活，以产生迅速增加的细胞体积，以补偿相邻细胞的损失和细胞生长。细胞体积和生长因子调节内皮细胞或其他细胞中NKCC1的机制尚不清楚。目前的证据表明，NKCC1是由磷酸化来调节的，但负责的激酶及其调节模式尚不清楚。这项提议的目的是证明在体外存在一个体积敏感的激酶，它能在体外磷酸化NKCC1，鉴定它，证明它在体内调节NKCC1，并证明该激酶受上游的体积敏感的激酶的调节，该激酶也受生长因子的调节。这将通过使用包含部分NKCC1的基因工程融合蛋白的体外激酶分析、亲和纯化以及体内NKCC1磷酸化和活性的分析来完成，在用反义寡核苷酸和显性的激酶缺失突变体特异性地抑制Kase之后。这些结果将提供关于内皮细胞中这一重要转运蛋白的功能和调控的重要信息，并确定受细胞体积和生长因子双重控制的特定调节激酶途径。

The objective of this proposal is to determine the molecular regulation of the Na-K-2Cl cotransporter (NKCCl) in vascular endothelial cells. This transporter is activated by cell shrinkage and rapidly restores cell volume, thus playing a potentially important role in minimizing gap formation in the endothelial monolayer and thereby maintaining the integrity of the blood-tissue barrier. The transporter is also activated by growth factors to produce rapid increases in cell volume necessary to compensate for loss of adjacent cells and for cell growth. The mechanism by which cell volume and growth factors regulate NKCCl in endothelial cells or other cells is unknown. Current evidence suggests that NKCCl is regulated by phosphorylation but the identity of the responsible kinase and its mode of regulation is unknown. The aims of this proposal are to demonstrate the existence of a volume-sensitive kinase that phosphorylates NKCCl in vitro, identify the kinase, prove that it regulates NKCCl in vivo, and to demonstrate that the kinase is regulated by an upstream, volume-sensitive kinase that is also regulated by growth factors. This will be accomplished with in vitro kinase assays using genetically engineered fusion proteins encompassing portions of NKCCl, affinity purification, and in vivo assays of NKCCl phosphorylation and activity after specific inhibition of kinases with antisense oligonucleotides and dominant kinase-deficient mutants. The results will provide important information on the function and regulation of this important transporter in endothelial cells and define a specific regulatory kinase pathway that is under dual control by cell volume and growth factors.